In the 55 years since the molecular defect responsible for sickle cell disease (SCD) was discovered, researchers have searched for anti-sickling agents to prevent the complications of the disease, but with little success. However, the total number of different chemical compounds that could have been evaluated over this period must be relatively small, due the limitations of the available technology. Recently, automated high throughput screening (HTS), has made it possible to rapidly screen libraries of hundreds of thousands of different small molecules to find promising drug candidates or possible new targets for drug development. At present, there are no assays for direct-acting anti-sickling agents that are well-suited for HTS. Assays that use hemoglobin S solutions are simple and amenable to automation, but do not address drug uptake or any other possible targets in the RBC, while morphologic sickling assays with intact RBCs are slow and difficult to automate and standardize. The aim of this study is to develop a simple and robust HTS compatible 384-well screening assay based upon a simplified model of vaso-occlusion. The assay measures the trapping of deoxygenated RBCs in the narrow channels formed between the beads in a Sephacryl column, and has a simple and stable endpoint that is read by optical imaging. The primary screen will detect whether the RBCs are trapped (the negative result) or pass through the gel (a positive "anti-sickling" result). Secondary assays will measure the activity (dose-response) and examine the mechanisms of action for each "hit". The phases of development will be: Examination of the contribution of all important assay variables, in particular, the influence of variation in the test RBCs; developing optimal assays and protocols that are robust, reproducible and sensitive; development of quality control procedures to insure reproducible performance of the test RBCs; and finally testing and further refinement of the assays and procedures during a semi-automated screen of >1500 compounds to simulate the use of the assays in a HTS environment.